Scanning ion conductance microscopy using surface roughness for probe movement

ABSTRACT

A method for interrogating a surface using scanning ion conductance microscopy (SICM), comprising the steps of:
         a) repeatedly bringing a SICM probe into proximity with the surface at discrete, spaced locations in a region of the surface and measuring surface height at each location;   b) estimating surface roughness or other characteristic for the region based upon the surface height measurements; and   c) repeatedly bringing the probe into proximity with the surface at discrete, spaced locations in the region, the number and location of which is based upon the estimated surface roughness or other characteristic in the region, and obtaining an image of the region with a resolution adapted to the surface roughness or other characteristic.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/953,122, filed Jul. 29, 2013, which is a continuation of U.S.application Ser. No. 12/864,302, filed Jan. 18, 2011, which is theNational Stage of International Application Number PCT/GB2009/050092,filed Feb. 2, 2009, each of which is hereby incorporated by referenceherein in its entirety, including any figures, tables, nucleic acidsequences, amino acid sequences, or drawings.

FIELD OF THE INVENTION

This invention relates to scanning ion conductance microscopy, and itsuse in the study of soft surfaces and interfaces, including those ofcells and convoluted matrix structures.

BACKGROUND TO THE INVENTION

Soft surfaces are a feature of many natural phenomena, particularly whenimmersed in liquid, including cell membranes and immiscible liquiddroplets. Many imaging and measurement techniques used for the study ofsuch surfaces employ a probing method that applies forces which mayinduce errors by disturbing the surface under observation or whichrequire modification of the surface before such observation can becarried out.

The cell is the most fundamental unit of living organisms, whetheranimal or plant. The study of its structure and composition, and how itsvarious constituents function, lends valuable insight into the complexprocesses that occur in integrated biological systems. This requirestechniques that allow investigation of cell samples to be conducted inreal-time, non-invasively, and in solutions that mimic physiologicalconditions so that cell functionality is retained.

Optical microscopy (using visible light) has been applied widely tostudy live cells. However, the resolution is limited by diffraction toabout 200-250 nm. For more detailed study, one commonly used method iselectron microscopy, where it is possible to obtain images with 10 nmresolution, but the sample needs to be fixed prior to imaging. Hence, itis not possible to use an electron microscope to study living cells.

Another possible high resolution technique is based on the use ofscanning probe microscopy (SPM), in which a sharp probe tip is scannedin close proximity to the sample under study. The consequentinteractions and thus the chemical/physical properties of the sample canbe plotted as a function of the tip's position with respect to thesample, to generate a profile of this measured interaction. Members ofthe SPM family that are commonly applied to biological imaging areatomic force microscopy (AFM), scanning ion-conductance microscopy(SICM) and scanning near-field optical microscopy (SNOM).

Atomic force microscopy (AFM) is commonly used to study the response ofa surface to mechanical force or pressure. When using AFM, the tipcantilever spring constant affects how much the surface under study willbe displaced by the measurement or detection process and sets a limit tothe softness of a surface which can be studied. An additional difficultywith AFM when used in contact or tapping mode is the likelihood of thesurface adhering to the probe tip, altering the measurements duringretraction and leading to contamination of the tip and mechanical damageto the surface.

In other cases the environment required by the probing method requiresmodification of the surface before imaging, as with electron microscopywhere the need for a vacuum or low pressure gas may requirestabilization of the surface and removal of liquid before imaging may becarried out.

Scanning ion conductance microscopy (SICM) is a form of scanning probemicroscopy (SPM) that allows the high resolution imaging of softsurfaces without any contact or force interaction whatsoever and in thenormal liquid environment of the subject. In SICM, typically anelectrolyte-filled, glass micropipette is scanned over the surface of asample bathed in an electrolytic solution; see Hansma et al (1989)Science 243:641-3. A quartz pipette may also be used. WO-A-00/63736discloses that SICM can be used effectively, e.g. to scan the surface ofa live cell by controlling the position of such a probe. This isachieved by adjusting the distance of the tip of the micropipette fromthe surface so as to maintain the current at a constant value, typicallythat which keeps the probe at a distance of some nanometers from it. Thepipette-sample separation is maintained at a constant value bymonitoring the ion-current that flows via the pipette aperture. The flowis between two electrodes: one inside the pipette and another outside inthe electrolyte solution. For an applied bias between the electrodes,the ion-current signal depends on a combination of the micropipette'sresistance (R_(P)) and the access resistance (R_(AC)) which is theresistance along the convergent paths from the bath to the micropipetteopening. R_(P) depends on the tip diameter and cone angle of themicropipette, whereas R_(AC) displays complicated dependence on thesample's electrochemical properties of the bath and the sample, geometryand separation from the probe. It is R_(AC) that lends ion-currentsensitivity to the pipette-sample separation and allows its exploitationin maintaining the distance such that contact does not occur.

The optimum tip-sample separation that has allowed SICM to beestablished as a non-contact profiling method for elaborated surfaces,is approximately one-half of the tip diameter; see Korchev et al (1997),J. Microsc. 188:17-23, and also Biophys. J. 73:653-8. The outputs of thesystem controlling the position of the tip are used to generate imagesof topographic features on the sample surface. The spatial resolutionachievable using SICM is dependent on the size of the tip aperture, andis typically between 50 nm and 1.5 μm. This produces a correspondingresolution.

The sensitivity of the micropipette used in SICM is highest to surfacesdirectly below the micropipette tip and is less so to surface structuresthat lie at the sides of the tip. If the micropipette is scanned acrossa surface whose features are of a similar scale to the diameter of themicropipette tip, the SICM system is able to keep the tip out of contactwith the surface. However, if the surface contains features andstructures of a height much larger than the tip diameter, and whichinclude steep edges or walls, then the scanning speed on the SICM systemmust be reduced in order to avoid collision, resulting in a longer timespent at each point on the surface. In the extreme, where the targetsurface is a convoluted structure such as a cluster of interwovenneurons, or a matrix or scaffold within which cells are growing, thereis a risk that the micropipette would become entangled and that the SICMsystem would be unable to scan.

WO-A-00/63736 discloses a method by which the probe is scanned acrossthe surface at a fixed speed or rate. Other methods are known in whichthe time spent by the probe at any point is varied to allow the probeion current and vertical position to stabilise within a given range. Inall of these methods, however, the resolution of the scan or density ofpoints measured during the scan, as represented by the number of pointsmeasured per scan line and the number of lines scanned per image, isconstant throughout that scan. As a result, an increase in the timespent at each point leads to a proportionate increase in the total scantime. The time to generate a high resolution image of a convolutedsurface may become so long as to be prohibitive.

Previous methods to confine the scan to the most relevant region havebeen based on optical microscopic surveys, including computer analysisof the resulting images (NASA/TM-2004-213383). This approach can becomplementary, but is limited to a resolution governed by the wavelengthof the light.

Mann et al, J. Neuro. Methods, 2003; 116: 113-117, discloses thetechnique of pulse-mode scanning ion-conductance microscopy. This isused to control the distance between the SCIM probe and the surface. Thetechnique uses constant current pulses to monitor the change inresistance.

Happel et al, J. Microscopy, 2003; 212: 144-151, discloses the use of apulse-mode scanning ion conductance microscopy to observe volume changesand cell membrane movements during the locomotion of cultured cells. Themicroscope apparatus uses current pulses to control the differencebetween the cell surface and the electrode tip as well as a back-step toprevent contact of the tip with the cell membrane during lateralmovements of the probe. The apparatus is used with a constant resolutionto determine areas having high surface structures. Lateral scans canthen be performed at different heights depending on the expected heightof the surface structures. Although this method has advantages, it stillresults in low scan speeds.

SUMMARY OF THE INVENTION

According to the present invention there is provided a method forinterrogating a surface using scanning ion-conductance microscopy(SICM), comprising the steps of:

-   -   a) repeatedly bringing a SICM probe into proximity with the        surface at discrete, spaced locations in a region of the surface        and measuring surface height at each location;    -   b) estimating surface roughness or other surface characteristics        for the region based upon the surface height measurements; and    -   c) repeatedly bringing the probe into proximity with the surface        at discrete, spaced locations in the region, the spacing of the        locations being based upon the estimated surface roughness or        other surface characteristic in the region, and obtaining an        image of the region with a resolution adapted to the surface        roughness or other surface characteristic.

According to a preferred embodiment of the present invention, the methodincludes sampling the surface to be scanned by SICM to ascertain whichareas are of most relevance to the investigator. The SICM micropipetteis cycled in height above the surface, with amplitude which is greaterthan the maximum hill valley distance, at discrete points across thesurface. To avoid collision or entanglement of the micropipette tipduring lateral movement between the discrete points, the micropipette ismoved laterally only while distant from the surface. No lateral movementof the micropipette which could damage the surface or tip takes placewhile the tip is close to the surface.

In the preferred embodiment the sampling takes place at four boundarycorner points for each square region of the surface. By analysis ofheight measurements from the initial four points, an estimate of surfaceroughness for the region can be determined. The scanning probe (e.g.SICM micropipette) is then cycled in height above the surface at anumber of points within the square, the number of points being selectedbased upon e.g. the estimated surface roughness. This analysis would beongoing for subsequent points to provide for a final scan over only theregion or regions with structures of interest, and within that region orregions to produce an image whose resolution is adapted locally to thesurface complexity.

Although surface roughness is described as the criterion for theadaptive scanning, to determine the number of pixels imaged in eachsquare on the surface, other criteria could be used depending on thebiological question of interest. For example, the presence of afluorescence signal could be used as a surface characteristic, toincrease imaging resolution.

Apparatus for enacting the method of the present invention comprises aSICM apparatus, including a scanning probe (micropipette), means formeasuring and/or controlling the distance of the probe tip from asurface to be scanned, and means for moving the probe laterally relativeto the surface.

In the preferred embodiment of the invention, the method includes thefollowing steps:—

-   -   (i) Optics and observation of change or modulation of the ion        current through the tip of the probe are used to bring the tip        to a controlled distance from the surface.    -   (ii) In repeated cycles the probe is retracted some distance        from the surface and then advanced back towards the surface at a        controlled velocity or rate.    -   (iii) During each of these repeated cycles, the ion current is        observed during the advance towards the surface until the        current falls below a preset threshold level, at which time the        probe is once again retracted.    -   (iv) To avoid sideways collision with surface structures, the        sideways motion control system is synchronised to the cyclic        advancement and retraction of the probe so that such sideways        motion is inhibited during the advancement part of the cycle,        and for the early part of the retraction.    -   (v) A region is selected and the probe is used to measure the        height of the surface at a few precursor points distributed        across that region. As already indicated, if the region is a        square, measurement could be made at the four corners of the        square.    -   (vi) The number of additional measurements to be made within the        region can be decided by analysing the distribution of heights        of the precursor points measured in Step (v). Where all such        precursor points are observed to have similar heights, it is        reasonable to assume that other points within the region will        also have similar heights, and that few or no further points        need to be measured. Where a significant variation in height of        the precursor points is observed, it may be appropriate to        measure the height of additional points distributed across the        region in order to determine the shape of the surface within the        region with greater fidelity. By not measuring at points        estimated to have similar heights and so reducing the total        number of points to be measured, the total time to produce an        image of the surface may be reduced significantly.

In the preferred embodiment, the height of the probe retraction in anyparticular region is determined taking into account an estimate of theheight range of structures in that region from observation of thevariation in heights of the precursor points, with the addition of asafety margin. By minimising the extent of retraction, the time requiredfor a full cycle of retraction and advancement is reduced, so reducingthe time taken to measure each point.

During the advance of the probe towards the surface, the motion of theprobe may be halted after the current falls below a preset threshold forthe first time, for a given time or for a given number of measurementsamples. Such additional measurements provide information about therelationship between ion current and distance from the probe to thesurface in the region close to that surface. The graphicalrepresentation of this relationship is commonly called an “approachcurve”. In turn, this relationship may indicate characteristics of thesurface, including its roughness, its conductivity relative to thesurrounding solution, or the degree to which it is normal to the probeaxis.

For each advance of the probe towards the surface, the threshold currentmay be set differently, taking into account the recent level of currentwhile the probe is distant from the surface. This current may bemeasured immediately prior to the advancement, may be an average of anumber of such measurements, or may be an average including suchmeasurements over a number of cycles of retraction. Such adaptation ofthe threshold may be necessary to take into account factors unrelated tosurface distance which might change the current.

Once points in a region have been measured, another region is selectedfor measurement. If this second region is adjacent to the first region,the data from any previous precursor points that lie on the boundarybetween these two regions are reused in estimating the height range ofthe second region. Thus superfluous measurement is avoided. In the caseof square regions, where the precursor points in the first region arefour corners, the estimation of height range for an adjacent squareregion would require the measurement of only two additional points.

Once the additional points in any region have been measured, an estimateis made of the height range in sub-regions within that region. If thatrange is large enough, a subset of the additional points is consideredas a new set of precursor points, and measurement made at further,additional points, more closely spaced than the first additional points,to determine the shape of the surface in that sub-region with greaterfidelity. This process may be applied recursively until a limitdetermined by the scanning resolution of the probe and the controllingsystem.

In a further aspect of the invention, the reduction in ion current asthe pipette (probe) approaches the surface is used to determineinformation about the surface curvature and mechanical properties of thesample at this point. The shape of the reduction in current containsinformation about both these additional properties of the surface, aswell as topography. If the surface is soft the pipette needs to movefurther down to get the same reduction in ion current since the surfacemoves away as the pipette approaches, due to forces exerted when thepipette is closer to the surface. The pipette also needs to move furtherdown if the surface is more curved. By analysing the approach curves andoptionally doing the approach at the same place at different appliedvoltages, which will alter the force applied, it is possible to obtainadditional information, and hence map these additional properties at thesame time. The present invention therefore makes it possible to mapother surface properties as well as the sample topography. This mayprovide more contrast in the obtained image making certain features ofinterest easier to detect, e.g. the underlying cell cytoskeleton underthe cell membrane.

In a further aspect, there is an apparatus for carrying out scanningprobe microscopy, comprising:

-   -   (i) a scanning probe;    -   (ii) means for measuring and/or controlling the distance of the        tip of the probe from a surface to be scanned;    -   (iii) means for moving the probe laterally relative to the        surface.        wherein the means for measuring and/or controlling the distance        of the tip of the probe comprises two piezo actuators of        different response times.

It is preferred if the first piezo actuator has a travel range of atleast 100 μm and the second piezo actuator has a travel range less than50 μm, preferably no more than 25 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the present invention will now be describedwith reference to the accompanying drawings, in which:

FIG. 1 shows the configuration of a hopping mode SICM system used toinvestigate a cell surface;

FIG. 2 illustrates the problem with conventional SICM, (b) illustratesthe hopping mode SICM to overcome the problem, (c) and (d) show scans ofhippocampal neurons with conventional raster scan (c) and hopping modeSICM (d), (e) illustrates the principles of hopping mode SICM;

FIG. 3 shows different scan resolutions for different regions of asurface;

FIG. 4 shows different compression levels for a region;

FIG. 5 shows the number of pixels in an exemplary prescan (A) and finalscan (B);

FIG. 6 shows images vertically protruding mechanosensitive stereoclia ofauditory hair cells, produced with hopping mode SICM;

FIG. 7 shows images of live hippocampal neurons;

FIG. 8 shows the configuration of a SICM system having primary andsecondary feedback systems;

FIG. 9 shows different compression levels utilised in the invention;

FIG. 10 illustrates probe movement during fluorescence mapping;

FIG. 11 shows images taken of live hippocampus neuron cells duringfluorescence mapping, where (A,D) are excited fluorescence SCIM imagesof neurons, (B,E) are differential fluorescence images between theexcited and reference fluorescence images of the neurons and (C,F) aretopographical SICM images of neurons; and

FIG. 12 is a simultaneous fluorescence signal recorded while scanning onlive hippocampus neurons, the arrow showing a burst in the signal whilethe pipette was held close to the cell membrane, which depolarised thecell membrane.

FIG. 13 shows the configuration of an SICM system of the subjectinvention.

DETAILED DESCRIPTION

A typical SICM apparatus of the invention comprises a scanning probe,piezo-actuator scanning elements, control electronics and a computer.These components may be built in and around an inverted microscope, e.g.Diaphot 200 (Nikon Corporation, Tokyo, Japan). The piezo-actuator can beused to measure and/or control the distance of the probe tip from thesurface to be interrogated and to move the probe laterally relative tothe surface.

The term ‘interrogate’ is intended to refer to the ability to monitorchanges at the surface of a structure, e.g. to detect structural changeson or at the surface at a single position or as the probe scans thesurface, or to measure the height of a structure. In certaincircumstances the surface may be pliable, and allow imaging ofstructures underneath the surface, e.g. cytoskeleton underneath a cellsurface. This is included in the term. It is not intended that the termbe restricted to detecting structural changes, and the monitoring of,for example, electrophysiological or chemical changes is also included.

The term “scanning ion-conductance microscopy” (SICM) is known in theart and relates to scanning probe microscopy whereby a probe ismaintained at a constant distance from a surface by the measurement ofconductance or resistance between the probe and the surface.

In an embodiment of the invention the software of an existing SICM unitis modified so as to drive the z-piezo (vertical) stage to implement thesequences outlined above. The estimate of the amplitude of thepreliminary “hopping” interrogation of the surface would be input by theuser, and is used by the software to govern the maximum z span of thepipette tip in approaching the initial points on the surface. The heightinformation derived from these probes is fed into the software tocalculate the positions of the next set of readings and so on until theregion of maximum roughness has been defined, when a normal scan isperformed in that region at the required resolution. The “hoppingamplitude” is usually greater than 1 μm, typically of the order 1 μm-8μm, more preferably 2 μm-6 μm, more preferably 3 μm-6 μm, e.g. 5 μm

The SICM, the pipette (probe) may be adapted such that, when located inproximity to the surface under study, a localised and controlledpressure or force can be applied to the measurement surface by means ofa regulated flow of liquid through the probe. The application of thispressure can be used to measure the flexibility or elasticity of thesurface by monitoring the relationship between the applied pressure andthe resulting movement of the surface. It can also be used to stimulatecell surface components, e.g. mechanosensitive ion channels, withsubsequent measurement of this stimulation carried out by monitoringconsequent changes in electrophysiological or chemical signals.

The pressure applied to the surface will, if the surface is sufficientlypliable, cause the surface to move. Positive pressure, i.e. flow throughthe probe towards the surface, has the effect of pushing the surfaceaway from the probe, increasing the separation between the surface andprobe tip. A negative pressure draws the surface towards the probe tip,decreasing the separation. The relationship between the applied pressureand the resulting movement of the surface can therefore provideinformation on the elasticity of the surface structure.

The probe may be used to scan the surface while simultaneously applyingpressure to it. In this way, the invention can be used to build up adetailed picture of the surface as it responds to the applied pressure,to reveal surface and sub-surface structures.

SICM probe images a surface without contact, and it can be operated in amode that exerts negligible force on the surface. For micropipettes ofapproximately 100 MegaOhm resistance this is the case when the appliedbias to the electrodes is low. At higher applied bias the electric fieldat the tip of such a micropipette is strong enough that forces due toelectrotension in the surrounding media are no longer negligible and arerecognised to be strong enough to actuate the constriction of cellmembranes (C. Bae, P. Butler Biomech. Model Mechanobiol. 7; 379: 2008).However, this electrotension has the additional effect of exerting asmall repulsive force on media of lower static dielectric constant,repelling them from the region of high electric field around themicropipette tip. At low voltages, when the micropipette tip is far fromthe surface, it is able to image while exerting no such force. However,at higher voltages, or when the micropipette tip is closer to thesurface, the higher electric strength gives rise to this small forcethat repels the surface slightly. Therefore, the difference in thesedisplacements at each point in an image can be exploited to measure themechanical properties of the sample; its Young's modulus and hysteresisin its movement for example. When the surface is not flat these measuredcoefficients must be corrected for the local curvature of the surface.This correction is realised technically by measuring at each point therelative displacements between two set percentages of decrease in ioncurrent, first at low voltage where deviation from the expecteddifference can only be due to curvature in the surface, and then athigher applied electrode bias, where the deviation may be expected toincrease because of the electrotensive force pushing the surface awayslightly. Moreover, this force can be exploited to constrain thesuperficial surface against elements underlying it so that these canalso be imaged by the micropipette.

The invention can be used to make a simultaneous measurement of astructure when the pipette is far from the surface and when it is closein the “hopping mode” so a differential map of the surface can beobtained by subtracting these two measurements taken shortly after oneanother. When the pipette is close to the surface it will locallydeliver an agonist or ions efficiently to the surface and give a largerresponse, measured by fluorescence detecting intracellular calcium orwhole cell recording of increased calcium channels, the delivery isnegligible when it is far away. Alternatively, the pipette can bestopped at the bottom of the hop and apply a voltage pulse (e.g. a short500 mx-2V pulse) for efficient local delivery—this differential mode canbe used to map receptors or mechanosensitive ion channels as outlinedbelow.

FIG. 10 illustrates the fluorescence mapping embodiment. The SCIM probefirst measures the background fluorescence signal while the probe isdistant to the surface waiting for lateral movement. Next, it approachesthe surface and measures the surface topography. The control softwaremay drive the probe (pipette) to jump up a small distance in order toprevent any further piezo movement. The feedback control is on hold andthe software measures increased fluorescence signal.

The apparatus can be adopted for simultaneous measurement of celltopography and detection of a fluorescence signal that is excited by alaser beam 5 from laser 10 focused at the tip of the pipette. Incontrast to existing technology, this “surface confocal imaging” can bedone by using the SICM in a “hopping mode”. At each “hopping” step, thelevel of background fluorescence will be recorded first when the pipetteis far from the surface and no force is exerted on the cell. The pipetteis then brought to a pre-defined distance from the surface of the celland both position of the pipette and fluorescence signal will besimultaneously recorded. When a pressure stimulus is applied to thepipette, it may activate mechanotransducer channels that are generallypermeable to calcium. This will give rise to increased fluorescence of acalcium-sensitive fluorophore 7 in the cell, such as Fluo-4. This signalcan be subtracted from the fluorescence signal when the pipette is faraway from the cell surface. Subtracting the background fluorescence willreveal the local changes in fluorescence on application of pressure.This technique will be used to map the position of, for example, MS ionchannels in DRG neurons and auditory hair cells.

Accordingly, the “hopping mode” combined with simultaneous fluorescenceor whole cell recording or any other simultaneous measurement of thesample can be used to obtain differential maps, map receptors andmechanosensitive ion channels and other properties e.g. local mechanicalproperties or local chemical groups. Multi-dimensional imaging ofsurface topography can be achieved and local surface properties orfunction probed by any agent or stimulus 9 delivered from the pipette.Measuring the response of the surface from this local change with thebaseline level of the parameter of interest measured just before orafter the bottom of the hop, provides a difference image. This techniquecould be used in a method to determine how drug candidates affect areceptor or mechanosensitive ion channel response or any agent whichalters the response.

In the case of an SICM probe adapted to apply a localised and controlledpressure, the hollow micropipette or nanopipette can be fabricated bypulling borosiliate glass capilliaries with, for example, outer andinner diameters of 1.00 mm and 0.58 mm respectively, using a laser-basedmicropipette puller (for example model P-2000, Sutter Instrument Co.,San Rafael, Calif., USA). Probes with conical taper lengths and apexdiameters of 200 nm, 400 nm and 1.0 μM, can be achieved. The pressurecan be exerted by conventional means to control the flow of liquidthrough the probe. Typically, a programmable pressure injector system,for example model PM-4, Warner Instruments, Hamden, Conn., USA, iscoupled to the shank of the SICM pipette holder by means of a flexibletube, and the injector programmed to generate the required pressure/timeprofile. The amount of pressure required can be determined by theskilled person. Typically, a positive pressure of at least 10 kPa, e.g.from 10 to 50 kPa is applied. More typically a pressure of from 13 to 40kPa is applied. The apparatus may further include means to measureelectrophysiological or chemical signals which may be generated by acell or biological surface and stimulated by the applied pressure. Suchmeasurement means is conventional in SICM.

The basic arrangement of an SICM for topographical imaging of livingcells has been described previously (Korchev et al., Biophys. J. 1997a;73:653-8; Korchev et al., J. Microsc. 1997b; 188 (Pt 1):17-23). Briefly,the SICM uses a patch-clamp nanopipette arranged perpendicularly to thesample as a scanning probe. The pipette is mounted on a three-axis piezotranslation stage.

In a traditional line scanning mode the probe approaches the cellsurface and scans over it while maintaining a constant tip-sampleseparation distance, using the SICM feedback control that keeps the ioncurrent through the pipette constant. The SICM controller produces asurface image of the cell, and makes possible a straightforward pipetteapproach precisely over an identified specific area or structure of thecell, to within approximately 100 nm from the cell membrane.

Whilst traditional line scanning SCIM can be used in both contact andnon-contact modes, the contact approach can be of limited use in cellmicroscopy, as the cells can become easily damaged, or the patch-clamppipette moved, breaking the high resistance patch due the directphysical contact, and/or disturbance by mechanical stimulation. However,non-contact mechanical stimulation could potentially be repeatedindefinitely, as there is no physical contact between the SICM probe andthe cell, avoiding damage.

Traditional SICM line scanning can take up to several hours to acquirehigh resolution images over a large area. Lengthy scanning works finewith fixed samples, however it is not applicable to living biologicalcells which change their surface formation from time to time. Drifts andstitch effects have been observed between adjacent areas over thescanned living cell surface in images obtained. The long scan time isprimarily due to the fact that the same high scan resolution is usedthroughout the image despite large portions of the scan area of thesubject surface being flat, or containing no interesting features. Thepresent invention advantageously reduces the scanning time by spendingless time to scan un-interesting features and using the most time toscan interesting areas.

In a preferred embodiment, the apparatus used to carry out the methodcomprises a second piezo-actuator, to improve the stopping ability ofthe probe.

There is a physical limitation for the time response of piezo actuatorsassociated with their travel range. Actuators with longer travel rangeare slower and not capable of faster settling times that can be achievedby shorter travel actuators of the same design. This behaviour isassociated with actuator resonant frequency that also drops with travelrange. For example a P-753 12 micrometer travel stage has 5.6 kHzresonant frequency, while the 38 micrometer travel version has only 2.9kHz (Physikinstrumente, Germany).

The hopping probe protocol requires immediate stoppage and withdrawal ofthe pipette when ion current is reduced to a specified set point.Delaying the stoppage and subsequent pipette withdrawal may result inpipette and/or sample damage due to collision.

A delay may be due to delayed vertical actuator response. In addition,momentum contributes to response delay taking it longer to stop withhigher fall (approach) rate therefore making faster imaging difficult.In order to overcome this, an additional piezo actuator of shortertravel range may be used, as shown in FIG. 1. The additional actuatorhas a faster response time in order to stop and withdraw the probe fastenough to allow ion current to recover to the initial (reference) valuewithout excessive drop. This should minimize the chances of collisionwithin the surface and enable faster imaging while retaining a longvertical travel range that is necessary to cope with tall samples. FIG.1 is a schematic diagram of the scanner where two piezo actuators arecombined in order to serve as one long travel high speed actuator.

Although the combined vertical actuator presented in FIG. 1 is intendedto be operated in hopping probe mode it can be used for conventional DCor modulated scanning.

Using this configuration, there is no prolonged excessive ion currentreduction. This is achieved by efficient withdrawal of the probe by thefast short-travel range piezo-actuator. The fast piezo receives a pulseof driving signal that is generated by hopping probe control softwareimmediately after the ion current reaches a specified set point.

If a combined vertical actuator system is used in DC or modulatedscanning, two separate feedback controls are used. Primary feedback thathas ion current as an input signal works as a SICM standard feedback andhas its output connected to drive a fast short-travel piezo-actuator.The secondary feedback uses a fast piezo-actuator position as an inputsignal and by adjusting the long travel actuator position brings thefast actuator to its middle range (see FIG. 8).

It is also possible to use the combined vertical actuator in the hoppingprobe mode when, at every measurement position, the system does notsimply acquire one height measurement with subsequent withdrawal of theprobe, but rather stops the long travel actuator and allows some timefor the fast piezo actuator to feedback and perform a more accuratemeasurement of height. This hybrid hopping probe and standard continuousfeedback mode allows higher vertical accuracy to be achieved.

In a preferred embodiment, the present invention uses a scanningprotocol developed to scan a subject surface with multiple resolutions.It scans interesting features with high resolution (slower scan) and lowresolution (quicker scan) for un-interesting ones. FIG. 3 shows an imagegenerated according to the principles of the present invention in whichthe flat, un-interesting region in the lower portion of the image hasbeen scanned at low resolution, whereas the rough, interesting region inthe upper portion of the image has been scanned at high resolution. Whencompared with traditional line scanning, a reduction in scan time of atleast 50 percent can be achieved using the present invention. Thistherefore allows cells to be imaged at higher resolution than haspreviously been possible.

Square Scanning

Instead of traditional line scanning, the entire surface to be imaged isdivided into a number of individual squares. These squares can each beimaged at different resolutions and so the resultant image has multipleresolutions. The square size is in pixels where each pixel is also usedas an imaging point. The square sizes used were 4×4, 6×6, 8×8, 16×16 and32×32 pixels. The image size is fixed to 512×512. The entire surface tobe imaged is scanned by imaging individual squares one by one. A 32×32square forms 16 squares in a row and 16 lines to give 256 squares in thewhole image. If a 4×4 square is used this yields 128 squares in a rowand 128 lines, giving 16384 squares in the whole image.

Compression Level

Compression levels determine imaging resolutions. Higher compressionlevels give higher compression and therefore lower resolution. However,it takes less imaging points and less scanning time. The compressionlevels used are: 1, 2, 4, 6, 8, 16 and 32. These levels are also inpixels; compression level 1 means image every pixel; level 4 meansimaging 1 pixel for every 4 pixel. A square cannot have compressionlevel higher than its side length. A 32×32 square can use anycompression levels up to 32, whereas a 4×4 square can only usecompression levels up to 4. For instance, if a 4×4 square is used asshown in FIG. 4, compression level 1 scans every pixel in the square andprovides 16 image points; compression level 2 scans every 2nd pixel inthe square and provides 4 image points; compression level 4 scans every4th pixel in the square and provides 1 image point. Reducing compressionby one standard level doubles the resolution but quadruples the durationof scan. Introduction of additional “intermediate” compression levels(see FIG. 9) between each pair of the standard compression levels allowsfiner control over the resolution and scanning speed. An intermediatecompression level d/√{square root over (2)} is created by overlaying twogrids of measurement points corresponding to compression level d,displaced one from another by d/2 pixels in both x and y direction (seeFIG. 9). Intermediate compression level d/√{square root over (2)} offers√{square root over (2)}-times higher resolution than the closest higherstandard resolution level at only 2-times slower scanning speed.

Compression Scanning Protocol

For every square, scanning consists of a prescan and a final scan. Theprescan is used to determine the resolution of the square, and thesubsequent final scan records the topographical data of the square.

Prescan

In the prescan, the probe is driven by the SCIM controller andinterrogates each of four corner pixels of the square in turn, as shownin FIG. 5A. The computer is configured to process the probe signals andcompare the subject surface height (z) values for each of the fourcorner points. The maximum difference between these four pixels iscompared with a defined “roughness” z height threshold. If thedifference is greater than the threshold then the square region boundedby the corner points is rough and so a high resolution final scan is tobe used, otherwise low resolution is used.

Final Scan

The final scan records topographical data after the prescan. The finalscan images the same square region at a number of discrete positions.The spacing between the final scan imaging positions is dependent on thecompression level elected by the computer following the prescan. As canbe seen from FIG. 5B, the square imaged at the four boundary corners inFIG. 5A is imaged in a final scan at 16 positions. The differentcompression levels used to scan the regions of the subject surface tocollect the topographical data represent the image resolutions of theresultant image. It is also possible to use only a single compressionlevel, such that the same resolution is used everywhere in the image.

The following Examples illustrate the invention.

Apparatus and Methods Used

Solutions

The standard external solution used for scanning of hippocampal neurons(Example 2) contained (mM): NaCl 145; KCl 3; CaCl₂ 2.5; MgCl₂ 1.2;Glucose 10; HEPES 10. The loading solution used for FM1-43 staining ofsynaptic boutons contained (mM): NaCl 103; KCl 45; CaCl₂ 2.5; MgCl₂ 1.2;Glucose 10; HEPES 10 and 10 μM FM1-43 (Molecular Probes). PBS(composition, in mM, NaCl 137, KCl 2.7, KH₂PO₄ 1.5, Na₂HPO₄ 4.3, pH 7.2)was used as external solution for high resolution imaging of the fixedcultured organ of Corti explants. Nanopipettes were filled with PBS inall experiments. Both the external and pipette solutions were filteredusing sterile 0.2 μm Acrodisc Syringe Filters (Pall Corporation, USA) tominimize blockage of nanopipettes during imaging.

Cultured Organs of Corti (See Example 1)

Organ of Corti explants were dissected from mice at postnatal days 2-4(P2-4) and placed in glass-bottom Petri dishes (WillCo Wells,Netherlands). The explants were cultured in DMEM medium supplementedwith 25 mM HEPES and 7% fetal bovine serum (Invitrogen, Carlsbad,Calif.) at 37° C. and 95% air/5% CO₂. Cultured organs of Corti were usedin experiments within 1-5 days. In some experiments, 10 μg/ml ofampicillin (Calbiochem, La Jolla, Calif.) was added to the medium. Theorgans of Corti from left and right cochleae of a mouse were processedsimultaneously. The cultured organs of Corti were immersed in 2.5%glutaraldehyde in 0.1M cacodylate buffer supplemented with 2 mM CaCl₂for 1-2 hours at room temperature. One cochlea was used for HPICMimaging while the other one was used for SEM imaging.

Hippocampal Neurons Preparation (See Example 2)

Hippocampal neurons were prepared as described in Shah et al, J.Neurophysiol. 2000; 83:2554-2561, and cultured on glass coverslips toallow confocal microscopy. Cells were kept in an incubator at 37° C. and95% air/5% CO₂ for 1 to 2 weeks. Once out of the incubator, cells werewashed with standard external solution and scanned within two hours, atroom temperature. For combined topography/fluorescent measurementshippocampal neurons were first incubated for 90 seconds at roomtemperature in 1.5 ml of loading solution to stain synaptic boutons withFM1-43 and then washed three times with a total volume of at least 10 mlof standard external solution and left for 15 min in the dark beforeimaging.

HPICM Probes

Nanopipettes were pulled from borosilicate glass (O.D. 1 mm, I.D. 0.58,Intracell, UK) using a laser-based puller Model P-2000 (SutterInstruments Co., USA). Two different pipettes were used: Standardpipettes, displayed resistances ranging from 100 MΩ to 150 MΩ (measuredin a standard external solution) and inner diameter of ≈100 nm. Thesepipettes were used for scanning hippocampal neurons (Example 2). Highresolution images of stereocilia bundles in cochlear hair cells(Example 1) were recorded with sharp pipettes, with resistances of ˜400MΩ (range 300-500 MΩ) and estimated inner diameter of ≈30 nm. Thepipette inner diameters are estimated from the pipette resistance usinga half cone angle of 3°.

Instruments

The hopping technique requires careful damping of mechanical vibrationsthat arise from the large, rapid vertical motions of the Z-piezo withthe attached probe. The apparatus has the nanopipette moving in theZ-direction while the sample is mounted on a separate piezo systemmoving it in the X-Y plane (FIG. 1). This separation of Z-piezo from theX-Y piezos is required to prevent mechanical interference. The circuitthat drives the movement of the piezo along the Z-axis is then tuned toallow a non-oscillating step response as fast as 1 ms.

All experiments were performed using a SICM scanner controller(Ionscope, UK) and scan head (Ionscope, UK). Two different heads wereused for imaging (FIG. 1). Scan head #1 consisted of a PIHera P-621.2 XYNanopositioning Stage (Physik Instrumente (PI), Germany) with 100×100 μmtravel range that moved the sample and a LISA piezo actuator P-753.21C(PI, Germany) with travel range 25 μm for pipette positioning along theZ-axis. Coarse positioning was achieved with translation stagesM-111.2DG (XY directions) and M-112.1DG (Z-axis) (PI, Germany). The Zpiezo actuator was driven by a 200 W peak power low voltage PZTamplifier E-505 (PI, Germany), while the XY nanopositioning stage wasdriven by 3×14 W amplifier E-503 (PI, Germany). Scan head #2 consistedof a P-733.2DD Ultra-High-Speed, XY Scanning Microscopy Stage (PI,Germany) customized for 10×10 μm travel range (XY movement of thesample) and a LISA piezo actuator P-753.21C customized for 5 μm travelrange (PI, Germany) that moved the nanopipette along Z-axis. Atranslation stage M-112.1DG with a travel range of 25 mm (PI, Germany)was used for coarse positioning of the pipette in the Z-axis. All piezoswere driven by 200 W peak power low voltage PZT amplifiers E-505 (PI,Germany). Scan head #2 was used for high resolution scanning of thecochlear hair bundles. All other experiments were performed using scanhead #1.

All piezo elements in both scan heads were operated in capacitivesensor-controlled closed-loop using Sensor & Position Servo-ControlModule E-509 (PI, Germany). Scan heads were placed on the platform ofinverted Nikon TE200 microscope (Nikon Corporation, Japan). The pipettecurrent was detected via an Axopatch 200B (Molecular Devices, USA) usinga gain of 1 mV/pA and a low-pass filter setting of 5 kHz. The internalholding voltage source of the Axopatch-200B was used to supply a DCvoltage of +200 mV to the pipette. The outputs of the capacitive sensorsfrom all three piezo elements were monitored using Axon Digidata 1322A(Molecular Devices, USA) and Clampex 9.2 (Molecular Devices, USA).

The LCS-DTL-364 laser diode (473 nm wavelength, Laser Compact, Moscow,Russia) was used to provide the excitation light source during confocalmicroscopy measurements. The fluorescence signal was collected usingoil-immersion objective 100×1.3 NA, an epifluorescent filter block and aphotomultiplier with a pinhole (D-104-814, Photon TechnologyInternational, Surbiton, England).

Hopping Mode Protocol

The vertical Z positioning of the hopping probe and the movement of thesample in the XY plane were controlled by a SICM controller (Ionscope,UK) utilising a SBC6711 DSP board (Innovative Integration, USA) at asampling frequency of 20 kHz. The measurement of height at each imagingpoint consisted of three phases. First, the probe was withdrawn from itsexisting position either by a specified distance or to a specifiedabsolute height level. Second, the vertical position of the probe wasmaintained for 10 ms, while the XY Nanopositioning Stage completed thesample's movement to a new point in the XY plane. During this time areference current I_(REF) was measured as an average of the DC currentthrough the HPICM probe. Finally, the probe was lowered at constant fallrate of 100 nm/ms (for a standard pipette) or 30 nm/ms (for a sharppipette) while monitoring the difference in current, ΔI, between I_(REF)and the instantaneous value of current through the probe I_(MV). As soonas ΔI exceeded the specified value of the setpoint, I_(S), during atleast four consecutive sample periods (that is 200 μs), the verticalposition of the probe was saved into the corresponding image pixel andthe probe was quickly withdrawn by a specified hop amplitude to start anew measurement cycle. I_(S) values ranged from 0.25 to 1% of I_(REF).

See “Approach curves” below for details on current measurement precisionand vertical resolution.

During adaptive imaging, the final topography of a “512×512” pixelsimage was actually acquired in squares of different sampling/resolutiondepending on the roughness observed within each square. The sizes of thesquares were 4×4, 8×8, 16×16, and 32×32 pixels, while the resolutionlevels were equivalent to 512×512, 256×256, 128×128, 64×64, 32×32, 16×16pixels per whole image.

One or two different resolution levels were used, for the images in thisstudy. In each square, a quick pre-scan at 4 corner points (FIG. 2e )was performed using a specified hop amplitude of 3 to 6 μm to determinethe roughness, R_(PP), and highest point H_(max). Each square was thenre-scanned at a higher resolution level if the estimated R_(PP) exceededthe user defined roughness threshold, R_(T), otherwise the lowerresolution level was used. In some cases, we simply imaged all squareswith the same level of resolution either to produce a fast preview image(at a low resolution) or to ensure that no details were lost duringadaptive scanning (a high resolution control). Large area images ofhippocampal neural network at a high resolution (Example 2) weretypically taken with a pre-scan hop amplitude of 5 μm, square size of4×4 pixels, two resolution levels of 256×256 and 128×128 pixels andR_(T) value of 100 nm. The pre-scan hop amplitude was reduced to 3 μmand the R_(T) value to 25 nm for the high resolution scans of hairbundles (Example 1). For medium resolution imaging of dendritic networks(Example 2) the square size was typically increased to 8×8 pixels andthe resolution levels of 128×128 and 64×64 were used. The overall timerequired to image the specimen varied significantly depending on theproportion of the sample area exhibiting high roughness. Generallyspeaking, imaging duration increased with slower probe fall rates (i.e.with sharper probes), smaller size of scan squares, higher pre-scan hopamplitude and higher resolution level. High resolution images ofelaborate samples took between 30 and 40 min. The images of lesselaborated areas took 5 to 12 min. Note that at each imaging square theupward hopping typically starts at the different initial heights.Therefore, the algorithm allows us to “climb” up a tall sample withoutan excessive increase of the amplitude of the hops in each imagingsquare.

Fluorescence Measurement

After being kept for 15 min in the dark, dishes with hippocampal neuronswere placed onto the XY nanopositioning stage in the scan head. Using an10× objective and X, Y and Z translation stages for coarse movement, theHPICM pipette was positioned over the region of interest and lowereddown to a safe distance of about 200 μm from the sample surface. Thenthe 100× oil-immersion objective was chosen and an automated approachalgorithm brought the HPICM probe to a distance of about one pipetteradius from the sample surface. The XY position of the whole microscopeplatform was then adjusted to align the tip of the pipette with theconfocal laser beam. To minimise photo-bleaching, fluorescence images ofthe selected area were recorded within 3 min separately from topography.The HPICM probe was retracted by ˜24 μm to prevent pipette—samplecollisions during rapid fluorescence acquisition. Topography imaging ofthe same area was performed immediately after obtaining a fluorescentimage.

Scanning Electron Microscopy

The fixed organs of Corti were dissected in ultra-pure distilled water,dehydrated in a graded series of acetone, and critical-point dried fromliquid CO₂. Then, the specimens were sputter-coated (EMS 575X SputterCoater, Electron Microscopy Sciences, USA) with 5.0 nm of platinum undercontrol with a film thickness monitor (EMS 150). The coated specimenswere observed with a field-emission SEM (S-4800, Hitachi Technologies,Japan) at low accelerating voltage (1-5 kV).

Image Processing

Raw height data obtained with varying resolution were interpolated usingbilinear interpolation to produce a final image of 512×512 pixels. Whenrequired, the images were corrected to remove stripes caused by smalldisplacement of XY nanopositioning stages in Z-axis and furthercorrected for the slope present in the preparations to aid visualisationof fine details.

Approach Curves

The experimental approach curve demonstrates that the minimum reliablydetectable current drop, expressed as a percentage of the referencecurrent recorded far from surface (I_(REF)), ranges from 0.25% (for astandard pipette of 100 nm inner diameter) to 0.75% (for a sharp pipetteof 30 nm inner diameter). Because of the high signal-to-noise ratio ofthe current measurements in our experimental setup, the estimatedvertical resolution at a 1% setpoint is 9 nm for a standard pipette and6 nm for a sharp pipette. The real vertical resolution depends on thelateral dimension of the feature. The vertical sensitivity of the 30 nmpipette can be further improved to ˜3 nm using a 1 kHz low-pass filterinstead of a standard 5 kHz. However, this would reduce the responsetime of the feedback control.

EXAMPLE 1

To determine the robustness of the technique, mechanosensitivestereocilia of the auditory hair cells in the cultured organ of Cortiexplants were imaged. Several attempts have been made previously toimage stereocilia with AFM or raster scan SICM, but these studies neverresolved even a gross structure of the stereocilia bundle. Specimenswere fixed to compare images obtained with the present invention(Hopping Probe Ion Conductance Microscopy—HPICM) and a scanning electronmicroscope (SEM) (FIG. 6a-c ). HPICM resolved stereocilia very well,including the shortest ones with a diameter of ˜100 nm or less (FIG.6b,c ). A kinocilium (true cilium), present in these young postnatalauditory hair cells, was also visualised (FIG. 6c , arrowhead). Toexplore the resolution limits of HPICM, fine extracellular filaments(links) that interconnect stereocilia and are crucial for theirmechanosensory function, were imaged. These links could be as small as˜8-10 nm in diameter. In wild type hair cells, most of the links areinaccessible to the HPICM probe, because it approaches vertically to thebundle. Therefore, we used abnormally short, but still mechanosensitive,stereocilia of Shaker 2 mice (FIG. 6d-f ). The HPICM probe, with aninner diameter of ˜30 nm, was able to resolve these links that appearedas features of 16±5 nm (n=37) in diameter (FIG. 6f ). HPICM uses thesame sensor as SICM and, therefore, shares the same physical principlesthat determine lateral and vertical resolution. The apparent diameter ofthe same stereocilia links on SEM images was 22±5 nm (n=41). Aftersubtraction of the platinum coat thickness (5 nm on both sides), anindependent estimate of 12±5 nm was obtained for the diameter of theselinks. The HPICM and SEM observations are therefore in excellentagreement, demonstrating the high resolution that is attainable.

EXAMPLE 2

Movements of live cells impose additional requirements for rapidimaging. To test whether adaptive HPICM is fast enough to visualize livecomplex cellular structures, live hippocampal neurons were examined(FIG. 7a ), which represent an unmet challenge for any scanning probemicroscopy because of the complex three dimensional shapes that areformed by axons and dendrites. HPICM revealed structures that resembledsynaptic boutons (FIG. 7b, c ) as well as very fine (down to 50-60 nm indiameter) processes, tentatively identified as axons (FIG. 7b,c ). Thisspecimen was labelled with FM1-43, an activity-dependent marker that isaccumulated in synaptic vesicles during cycles of endo- and exocytosis,and recorded the topography and the FM1-43 fluorescence of the samesample. Whenever a fluorescent signal was observed, it was also possibleto identify varicosities in the images (FIG. 7d-g ). The size and shapeof these varicosities is consistent with the geometry expected ofsynaptic boutons. It is thus clear that the speed of adaptive HPICM issufficient to generate a “snapshot” of axons, dendrites and boutons inthese complex live networks in spite of relatively slow (on a time scaleof tens of minutes) re-arrangement and migration of the cells that dooccur in this preparation. Faster dynamics can be followed by imaging ofa smaller area and/or decreasing the resolution.

EXAMPLE 3 Mapping of Ion Channels on Living Cells

Mapping ion channels on cell membrane has been a great interest inbiology. With hopping mode, live neurons were stimulated to open theirion channels by depolarisation of the cell membrane. The response ofneurons was measured using fluorescence detection with simultaneousscanning. The cells were loaded with fluo4 dye which is sensitive tohigh calcium concentration and the pipette was filled with solution thatcontained potassium. Potassium ions released from the pipette tipdepolarised the cell membrane, while the pipette close to the surface,causing the cell membrane to open its calcium channels. Calcium ionsentered the cell through these channels and combined with fluo4, causingthe dye to become fluorescent.

A negative voltage potential in the pipette was used to keep potassiuminside the tip prior to the scan. During scan, a positive voltage wasused for dosing potassium out of the pipette during scan. The hoppingsetpoint used was between 0.5%-0.7% drop of the reference ion currentand the hopping amplitude was between 5˜7 μm. At every image point, thepipette was positioned 80 nm above the cell membrane for 80 ms. In thisperiod, potassium ions released from the pipette locally depolarised thecell membrane and the excited fluorescence signal, due to influx ofcalcium, was collected as shown in FIGS. 11 A&D. In order to remove thebackground fluorescence, a reference signal measured when the pipettewas far away from the membrane at each pixel, was subtracted from theexcited fluorescence signal to produce a differential signal (FIGS. 11B&E). From these images, it can be clearly seen that part of neuron cellbody and dendrites were depolarised by the pipette's stimulation, andthe ion channels were mapped as a result. The excited fluorescencesignal was recorded and burst was observed while the pipette was closeto the cell membrane, which is indicated of the opening of ion channels,as shown in FIG. 12.

The content of all publications described above is incorporated hereinby reference.

We claim:
 1. A method for interrogating a surface using scanning ionconductance microscopy (SICM), comprising the steps of: a) repeatedlybringing a SICM probe into proximity with the surface at discrete,spaced precursor points in a region of the surface to obtain a precursorpoint surface height measurement for each of the precursor points; b)estimating surface roughness for the region by analyzing thedistribution of the precursor point surface height measurements; c)determining a number and location of discrete, spaced additional pointsbased upon the estimated surface roughness; d) repeatedly bringing theprobe into proximity with the surface at the additional points to obtainan additional point surface height measurement for each of theadditional points; and e) combining the precursor point and additionalpoint surface height measurements to obtain an image of the region witha resolution adapted to the surface roughness.
 2. The method accordingto claim 1, wherein steps (b) to (d) are repeated recursively forsub-regions according to the required image resolution.
 3. The methodaccording to claim 1, wherein the step of bringing the probe intoproximity with the surface at each precursor or additional point isperformed by approaching each precursor or additional point from adistance greater than the height of the surface at that precursor oradditional point.
 4. The method according to claim 1, wherein lateralmovement of the probe occurs only when the probe is distant from thesurface.
 5. The method according to claim 1, wherein, during the step ofbringing the scanning probe into proximity with the surface at eachprecursor or additional point, the approach is terminated when ameasured probe current reaches a threshold value.
 6. The methodaccording to claim 5, wherein the threshold value is based upon theprobe current measured when the probe is distant from the surface. 7.The method according to claim 5, wherein the approach is terminated whenprobe current is reduced by 0.25% to 1%.
 8. The method according toclaim 6, wherein for each measurement, the distance travelled by theprobe, from the position distant from the surface to the position at thethreshold value, is greater than 1 μm.
 9. The method according to claim1, wherein, during the step of bringing the scanning probe intoproximity with the surface, the approach rate or speed is constant. 10.The method according to claim 1, wherein a local relationship betweenprobe current and distance from probe to surface is determined for eachprecursor or additional point.
 11. The method according to claim 1,wherein a differential map of the surface is obtained by, for eachprecursor or additional point, obtaining a first scanning measurementwhen the probe is distant from the surface and a second scanningmeasurement when the probe is in proximity to the surface andsubtracting the second scanning measurement from the first, to obtainthe differential map.
 12. The method according to claim 11, wherein anagent or other stimulus is applied at the tip of the probe andmeasurements of response to the agent or stimulus are made together witheach first or second scanning measurement to provide a differential mapof the surface.
 13. The method according to claim 11, carried out in thepresence of a fluorophore the intensity of which is increased by asurface structure, wherein a laser beam is focused at the tip of theprobe to induce fluorescence, wherein a scanning measurement is obtainedtogether with each first or second scanning measurement, and whereinsubtraction of the second fluorescence measurement from the firstreveals local changes in fluorescence.
 14. The method according to claim11, wherein, during the step of bringing the probe into proximity withthe surface at each precursor or additional point, the approach isterminated, and an image is obtained, when a measured probe currentreaches multiple different threshold values.
 15. The method according toclaim 14, wherein the approach is terminated when probe current isreduced by 1%, 5% and 10%.
 16. The method according to claim 1, whereinsteps (b) to (d) are carried out using estimated surface roughness. 17.The method according to claim 1, wherein steps (b)to (d) are carried outby measuring the presence of a fluorescence signal.
 18. The methodaccording to claim 5, wherein an image is obtained at multiple differentthreshold values for measured probe current, where differences in theresults obtained provide information on the mechanical properties of thesurface or reveals information on structures underneath the surface. 19.The method according to claim 1, wherein step (d) is carried out atmultiple different voltages, where differences in the results obtainedprovide information on the mechanical properties of the surface orreveals information on structures underneath the surface.
 20. The methodaccording to claim 1, wherein the region is square, the precursor pointsare located at each of the square region's corners and steps (c) to (e)are performed following a determination that the maximum differencebetween any of the precursor point surface height measurements isgreater than a predefined roughness threshold.
 21. The method accordingto claim 1, further comprising estimating height range in sub-regionswithin the region and, if that range is large enough, considering asubset of the additional points as a new set of precursor points andmaking measurements at further additional points, more closely spacedthan the previously measured additional points.
 22. The method accordingto claim 21, wherein the further estimating height range, considering asubset of additional points, and making measurements steps are appliedrecursively until a limit determined by the scanning resolution of theprobe and its controlling system is reached.